SUMMARY OF WORK Our recent studies have shown that the beta2-adrenergic receptor (beta2AR)-stimulated increases in intracellular Ca2+ (Cai) transient and contraction in cardiac myocytes are dissociated from the increase in cAMP level and are not accompanied by an increase in phospholamban phosphorylation, or an acceleration of relaxation or a reduction in myofilament Ca2+ response. Thus, we hypothesized that the beta2AR modulation of cardiac excitation-contraction (EC) coupling may be mediated by either a cAMP-independent mechanism or a compartmentalized cAMP pathway. To directly distinguish these two possibilities, the responses of L-type calcium current (ICa), Cai transient and contraction to beta2AR as well as beta1AR stimulation were examined in rat ventricular myocytes in the presence or absence of specific inhibitory cAMP analogues, Rp-cAMPS (Rp) and Rp-CPT-cAMPS (Rp-CPT). As expected, the positive inotropic effect induced by an adenylyl cyclase activator, forskolin (2x10-7 M), or a beta1AR agonist, norepinephrine (5x10-8 M) plus prazosin (10-6 M) was completely blocked by Rp-CPT. More importantly, the responses of ICa, Cai transient and contraction to beta2AR stimulation by zinterol (10-5 M), or by isoproterenol plus a selective beta1AR antagonist CGP 20712A, were also entirely abolished by Rp (in the patch pipette solution) or Rp-CPT (in the bath solution). In pertussis toxin (PTX) treated cells, while the response of cAMP was not altered, the beta2AR stimulated increase in contraction amplitude was markedly enhanced and accompanied by hastened relaxation, resulting in a tight association between cAMP and contraction. These results indicate that beta2AR modulation of cardiac excitation- contraction coupling requires cAMP. The dissociation of beta2AR stimulated cAMP production and regulation of myofilament and sarcoplasmic reticulum functions is attributable to a functional compartmentation of the cAMP-dependent signaling due to an activation of beta2AR-coupled Gi/Go.